BACTERIA OF THE SOIL 61 



Exercise 88. Preparing Dilutions and Pouring Plates 



1. Weigh 1-g. samples of soil into 99-cc. water blanks. Stir 

 with a sterile glass rod until all lumps are broken up. 



2. Draw 1 cc. of the muddy water with a sterile pipette and 

 transfer to another 99-cc. water blank. These water blanks 

 should be contained in narrow-necked flasks or bottles of 200 cc. 

 capacity, to permit of agitation without loss of their contents. 

 Agitate for two minutes ; then transfer 1 cc. with a sterile pipette 

 to a sterile Petri dish and add a tube of melted agar which has 

 cooled to 43 C. Mix the contents by tilting the dish. When 

 cool, incubate at a temperature between 18 and 23 C. 



For making these plates agar media are preferable to gelatin, 

 on account of the higher nitrogen content of the latter. Beef- 

 peptone agar may be used, although the Heyden-Nahrstoff agar 

 will give higher counts. Lipman 1 reports good results from the 

 use of a synthetic agar. 



3. Count the colonies as they develop and estimate the num- 

 ber of bacteria per gram. 



4. Many of the bacteria of the soil are anaerobic and can 

 only be grown in the absence of oxygen. For method see 

 Exercise 56. 



Exercise 89. Determination of the Number of Spores in Soil 



1. Make a soil suspension in a dilution of 1 : 1000. Add 1 cc. 

 to a tube of sterile melted gelatin. 



2. Heat the tube in a water bath to a temperature of 80 C. 

 for ten minutes. 



3. Pour the melted gelatin into a sterile Petri dish. Incubate 

 at room temperature and count the colonies after four days. 

 Estimate the number of spores in 1 g. of the soil sample. 



i Lipman and Brown. Centralbl. f. Bakt., 2te AM., 25 : 447. 1910. 



Water 1000.00 cc. 



Dextrose 10.00 g. 



K 2 HP0 4 0.50 g. 



MgS(> 4 0.20 g. 



Peptone 0.05 g. 



Agar-agar 20.00 g. 



